Classical alkylating agents evidently produce their therapeutic as well as their mutagenic and carcinogenic effects by attacking the DNA of target cells. The newer nitrosourea compounds, bis-chloroethyl nitrosourea (BCNU) and chloroethyl cyclohexyl nitrosourea (CCNU), alkylate nucleic acids to a lesser extent, but the specificity of the reaction is different. Other agents such as procarbazine probably react in still different ways. The sites of base substitution will be determined and the significance of each base modification will be investigated in both DNA and RNA. Synthetic polynucleotides will be used as models for nucleic acids in these studies so that effects on particular nucleosides can be studied separately. Since these polymers are effective templates for replication by RNA polymerase, alkylated and unalkylated polymers can be compared to determine which reactions are biologically significant. By obtaining data on different classes of agents - e.g., classical alkylating agents vs. nitrosoureas - we will look for explanations of the clinically-observed differences among these agents. Some of the modified nucleosides which we have identified in the initial period of this grant are now ready for animal testing as antitumor agents in their own right. Thus, fluoroethyl cytidine and fluoroethyl guanosine, which are formed by the transfer of a fluoroethyl carbonium ion from BCNU to the corresponding nucleoside, will be tested for activity against P388 cells. Other derivatives will also be tested as they are identified and synthesized. These studies will serve the dual purpose of elucidating the mechanism of action of the parent compound and possibly uncovering new antitumor agents. Although major emphasis will be placed on the therapeutic value of these agents, attention will also be paid to possible mutagenic and carcinogeic side reactions.